Modifications of the chromatin arrangement induced by ethidium bromide in isolated nuclei, analyzed by electron microscopy and flow cytometry.

Ethidium bromide (EB) is widely used for investigating the DNA conformation in chromatin both with conventional and cytofluorimetric techniques. Since the interaction of the dye with DNA should result in structural deformations which can be different in isolated or in situ chromatin, a study has been performed on the effects caused by different amounts of EB and the analogous propidium iodide on isolated nuclei, in which chromatin maintains its native relationships with the other nuclear structures (envelope, nucleolus, interchromatin RNP, nuclear matrix). The results obtained by comparing ultrastructural observations in thin sections and in freeze-fracturing with conformational analysis in multiparameter flow cytometry indicate that the phenanthridinic fluorochromes, especially at the high concentrations used for cytofluorimetric analyses, cause deep rearrangements of the chromatin in situ.

Changes of chromatin organization induced by phospholipids.

Isolated nuclei represent a suitable model for studying the influence of exogenous phospholipids, normally found as minor chromatin components, on the nuclear structure, which, in turn, could be related to the observed modifications of DNA and RNA synthesis. The morphological modifications induced on chromatin RNP granules and nuclear matrix have been analyzed both with conventional thin sectioning and with an original method based on image analysis of freeze-fractured and replicated nuclear samples. The results obtained support the hypothesis that anionic phospholipids, by removing histone H1, induce a transition of the chromatin from solenoid to nucleosome conformation and favour the RNA polymerizing activity which results in an increased release of RNP particles, while neutral phospholipids, probably affecting the matrix structure, partly impare the RNP maturation and transport, with consequent increase of chromatin condensation.

Morphometric and topologic analysis of freeze-fractured interphase nuclei.

Interphase rat liver nuclei were studied by freeze fracturing followed by electron microscopic observations. This method permits information on the native organization of the nuclear components in the hydrated state to be obtained. Morphometric analyses, performed with a Leitz Texture Analysis System, gave precise information on the different nuclear components, based on the histograms of their size distribution in heterochromatin, interchromatin and nucleolar areas.

Electron microscopy microsampling of isolated nuclei sorted by flow cytometry.

This paper describes an efficient method to concentrate for electron microscopic examination minute quantities of subcellular particles obtained by cytofluorimetric sorting. The advantages of this micromethod, based on diafiltration on Millipore filters under constant positive nitrogen pressure, are discussed.

Flow cytometric analysis of liposome-nuclei interaction: transfer and intranuclear release of carboxyfluorescein.

Flow cytometric analysis of the transfer of liposome-encapsulated carboxyfluorescein to isolated rat liver nuclei indicated that the fluorophore is actively taken up in this form, while negligible amounts are transferred when the dye is free in the reaction medium. The kinetic analysis of the uptake indicated a time- and dose-dependent reduction of the slope in the absence of transport saturable sites on the nuclear surface and of quenching phenomena. The comparison between entire and membrane-deprived nuclei demonstrated that the initial rate of uptake was higher in the absence of the complete nuclear envelope.

Morphometric analysis and topological organization of nuclear matrix in freeze-fractured electron microscopy.

The ultrastructural organization of nuclear matrix, purified from intact or membrane-denuded rat liver nuclei, has been analysed by means of freeze-fracturing technique. This method avoids dehydration and embedding which, in conventional thin sectioning, partly distort or mask the matrix ultrastructure. The various matrix components, and mainly the peripheral lamina and the inner network revealed complex arrangements undetectable with conventional techniques.

Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial FoF1 H+ ATPase in regenerating rat liver.

A study of the FoF1 ATPase complex of mitochondria isolated from regenerating rat liver following partial (70%) hepatectomy is presented. As we have previously reported, ATPase activity in submitochondrial particles prepared from regenerating rat liver 24 h following partial hepatectomy was depressed by 75% with respect to controls (submitochondrial particles from sham-operated animals). Polyacrylamide gel electrophoresis and immunodecoration using an antibody raised against isolated bovine heart F1 sector of the FoF1 ATPase indicated a substantial decrease in F1 content in the mitochondrial membrane from regenerating rat liver.

Effect of 2,4-dinitrofluorobenzene on the enzymatic properties of the b-c1 complex isolated from beef heart mitochondria.

A study is presented on the effect of 2,4-dinitrofluorobenzene (DFNB) on the enzymatic properties of mitochondrial b-c1 complex. The chemical modification by DNFB strongly inhibits the reductase activity of the complex, this being accompanied by labelling by [3H]DNFB of core protein I, the apoprotein of b cytochromes and the 12 kDa subunit. Chemical modification by DNFB appears to alter, in particular, the domain of heme b-562.

Effect of phospholipids on transcription and ribonucleoprotein processing in isolated nuclei.

The response of isolated rat liver and murine erythroleukemia nuclei to phospholipid liposomes has been monitored with different techniques, by studying the endogenous RNA synthesis, the release of transcripts in the medium, the pattern of acid-extractable nuclear proteins and the ultra-structural morphology. Total transcription in rat liver and beta-globin mRNA synthesis in MEL nuclei are increased by PS and reduced by PC. These changes of RNA polymerase activity, and the transport of RNAs from nucleus as well as the nuclear protein changes, correlate with structural transitions which occur in both types of nuclei, consisting of euchromatization with loss of RNP particles in the case of PS and opposite effects with PC.

Ultrastructural organization of freeze-fractured interphase nuclei.

The morphology of intact or membrane-deprived interphase nuclei has been analysed by freeze-fracture electron microscopy. This method appears particularly useful for providing information on the distribution and organisation of chromatin and ribonucleoproteins in the absence of dehydration and embedding artifacts of conventional electron microscope techniques which, among other effects, appear to affect heterochromatin distribution, inducing its aggregation along the nuclear envelope. The main levels of chromatin superstructure, from nucleosome to solenoid fibres, are detectable in the replicas of freeze-fractured nuclei on the basis of the size of their shadow, a parameter particularly suitable for automated image analyses.

Effect of diamide on proton translocation by the mitochondrial H+-ATPase.

Treatment of sonic submitochondrial particles with the bifunctional thiol reagent, diamide, results in an enhancement of proton conductivity and ATPase activity, which is reversed by the reducing agent dithiothreitol, is suppressed by Fo inhibitors like oligomycin and is absent in particles that are deprived of peripheral Fo polypeptides. The effect of diamide is apparently due to oxidation of dithiols to disulfides in peripheral polypeptide(s) of Fo.

Changes in activity and F1 content of mitochondrial H+-ATPase in regenerating rat liver.

Submitochondrial particles prepared from rat liver during hepatic regeneration exhibit a depressed ATPase activity which is correlated with a decrease in F1 subunit content as shown by SDS-PAGE. Use of an antibody directed against the F1 portion of the H+-ATPase complex demonstrated that there is a definite decrease in the amount of beta-subunit of F1 in both submitochondrial particles and mitochondria from rat liver 24 h after partial hepatectomy.

Effect of papain digestion on redox-linked proton translocation in b-c1 complex from beef heart reconstituted into liposomes.

Papain treatment of the cytochrome b-c1 complex from beef heart results in partial proteolysis of core protein II, the iron-sulphur protein and the 15-kDa subunit. Under these conditions a significant inhibition of electron flow and complete suppression of proton translocation in the complex reconstituted into liposomes are observed. Kinetic experiments indicate a correlation between the digestion of core protein II and 15-kDa subunit and the suppression of proton translocation.

Characteristics and nature of redox-linked proton transfer reactions in cytochrome c oxidase of mitochondria.

A discussion is presented of the characteristics of proton transfer reactions associated to redox catalysis in mitochondrial cytochrome c oxidase. These properties are examined in the light of the mechanisms proposed for the conversion of redox energy into a transmembrane proton gradient. It is concluded that this energy transfer process is first of all due to the anisotropic arrangement of the reduction of oxygen to H2O in the oxidase.

Response of isolated nuclei to phospholipid vesicles: a two dimensional gel electrophoresis analysis of H2SO4 soluble nuclear proteins after treatment with phosphatidylserine.

H2SO4 soluble proteins extracted from nuclei incubated with phosphatidylserine multilamellar vesicles (PS MLV) have been analysed by means of two-dimensional gel electrophoresis with particular attention to the uH2A fraction. A reduction of H1, H1 degrees and proteins A5, B7, B15 and B23 has been observed in lipid treated nuclei, while the core histones, as well as uH2A are unaffected by liposome treatment. Since these proteins show in vitro the same binding affinity for PS, their behaviour appears to be related to difference in localization in the nucleosome, responsible for their variable accessibility in the chromatin.

Increase of globin RNA synthesis induced by phosphatidylserine liposomes in isolated erythroleukemic cell nuclei. Morphological and functional features.

The effect of phospholipid vesicles on chromatin structure, protein composition and globin RNA synthesis has been analysed in nuclei isolated from murine erythroleukemia cells. In terms of chromatin organisation, PC vesicles with neutral surface charge do not affect the structure of chromatin fibres, whereas negatively charged PS vesicles induce chromatin decondensation to a great extent. Indeed the fibres appear uniformly dispersed lacking also the perinucleolar heterochromatin.

Interaction of the aminoglycoside antibiotic dihydrostreptomycin with the H+-ATPase of mitochondria.

In this paper a study is presented of the effect of dihydrostreptomycin on the H+-ATPase of the inner mitochondrial membrane. The antibiotic caused at concentrations of 1-5 X 10(-3)M a marked enhancement of the hydrolytic activity of the H+-ATPase complex in intact mitochondria and submitochondrial particles which was accompanied, in the latter, by enhancement of passive transmembrane proton conduction by the complex. The stimulation by dihydrostreptomycin of ATP hydrolysis resulted in a suppression of the sensitivity of this activity to inhibition by oligomycin.

On the mechanism of H+ translocation by mitochondrial H+ -ATPase. Studies with chemical modifier of tyrosine residues.

In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H+ conduction and other reactions catalyzed by the H+ -ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-33Pi exchange, and proton conduction by the H+ -ATPase complex. These effects are caused by nitration of tyrosine residues of H+ -ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-33Pi exchange in the complex purified from tetranitromethane-treated particles.

Response of isolated nuclei to phospholipid vesicles: analysis of the nuclear proteins after treatment with phosphatidylserine and phosphatidylcholine and comparison with heparin.

Phospholipid liposomes affect the histone pattern of isolated rat liver nuclei. Multilamellar vesicles (MLV) obtained with phosphatidylserine (PS) release a large amount of the lysine rich histones, while those obtained with phosphatidylcholine (PC) do not induce significant changes with respect to controls. This different response has been compared to the effects obtained with Heparin, which slightly modifies the relative ratio of the histone fractions.

Transfer of liposome-encapsulated carboxyfluorescein to isolated nuclei.

The interaction between phosphatidylcholine vesicles and isolated rat liver nuclei has been examined by studying the uptake of the fluorophore carboxyfluorescein. The kinetics of transfer of the dye, analyzed by flow cytofluorimetry with a Fluorescence Activated Cell Sorter (FACS IV), indicate an efficient delivery to the nucleoplasm. The results reflect a liposome-nuclear membrane interaction which may contribute to the processes which underlie our previously described morphological and functional changes in isolated nuclei treated with phospholipids.

Distribution of acid alpha-naphthyl acetate esterase among human T lymphocyte subsets.

Human T lymphocyte subsets, identified by means of OKT3, 4 and 8 monoclonal antibodies, were isolated by a fluorescence activated cell sorter (FACS IV) and analyzed for distribution of alpha-naphthyl acetate esterase (ANAE) activity. As compared to OKT8+ lymphocytes a higher proportion of OKT4+ lymphocytes was ANAE-positive exhibiting a spot or dot-like pattern in the cytoplasm. OKT8 and 4 positive subsets showed a similar ANAE distribution in diffuse granular form.

Response of isolated nuclei to phospholipid vesicles: analysis of chromatin sensitivity to DNase I and micrococcal nuclease.

Phosphatidylserine (PS) and phosphatidylcholine (PC) multilamellar vesicles (MLV) affect chromatin structure as analysed by DNase I sensitivity. The kinetics of DNA solubilisation during the digestion of nuclei indicates that phosphatidylserine causes an increase in DNase accessibility while phosphatidylcholine slightly reduces this accessibility. The effect of phosphatidylserine has also been analysed by means of isokinetic sucrose gradients and agarose gel electrophoresis of nuclear DNA solubilised by micrococcal nuclease.